This invention concerns a novel chemical agent which significantly prevents or blocks non-specific staining or binding of an antibody specific for Terminal Deoxynucleotidyl Transferase (TdT) in biological specimens during immunofluorescent or immunoperoxidase assay procedures. More particularly, the invention employs a casein selectively introduced at an appropriate stage of the assay procedure prior to analysis using a TdT specific monoclonal antibody which has been tagged or labelled to enable either immunofluorescent or immunohistochemical staining of samples followed by flow cytometric and/or microscopic analyses, respectively.
TdT is a unique DNA polymerase associated with early T-and B Lymphocyte differentiation in humans, as well as other species. Although TdT is found in a very small percentage of normal lymphoblasts, particularly in the early development of the immune system of vertebrates, elevated levels of TdT have been used in the diagnosis of human leukemias. TdT has become a valuable enzymatic marker for lymphoblastic neoplasms, such as acute lymphoblastic leukemia (ALL), chronic granulocytic leukemia (CGL) and lymphoblastic lymphoma (LL). Consequently, research has been conducted to develop methods for measurement of the frequency of lymphocytes which are positive for TdT in both normal and leukemic mammals. U.S. Pat. No. 4,307,189 describes a method for quantitative determination of TdT using labelled deoxynucleoside triphosphates which are converted by TdT to fluorescent or radioactive polydeoxynucleotides which may be quantified as a reflection of the amount of TdT originally present in the biological sample. However, this method does not employ monoclonal antibodies to TdT.
In studies published by C. Augl et al (Fed. Proc. 42:2147 1983) (Abstract) the production of monoclonal antibodies to bovine TdT has been reported without description of detailed binding recognition of the antibodies. Immunochemical studies of TdT in a variety of mammals have demonstrated that peptides of this enzyme are immunologically related when probed with antiserum prepared to the degraded enzyme from bovine thymus as reported by F.J. Bollum (Journal of Biological Chemistry 256:8768, 1981).
In studies published by F.J. Bollum, et al. (Journal of Biological Chemistry 259: 5848, 1984), the production of monoclonal antibodies to human TdT has been described. These anti-human monoclonal antibodies were widely variable in ability to recognize epitopes or determinants on TdT in human and calf cells.
Coulter Corporation, the assignee of this patent application, has introduced into the market through its Coulter Immunology Division (CID) a TdT monoclonal antibody immunoperoxidase assay kit for research use. This assay enables investigation of lymphoblastic disorders, including acute lymphoblastic leukemia (ALL) and chronic myelogenous leukemia (CML) in blast crisis studies. The TdT monoclonal antibody supplied is conjugated to a label or tag, such as fluorescein isothiocynate (FITC), for direct immunofluorecent staining and analysis by flow cytometry or fluorescent microscopy. This conjugated TdT monoclonal antibody enables anaylsis and enumeration of TdT positive cells in normal and neoplastic hematopoietic tissue and blood.
The TdT monoclonal antibody utilized in said assay kit of CID was developed by R. Graham Smith, a co-inventor named in this patent application. This monoclonal antibody is one of several developed by R. Graham Smith which are specific to a unique antigenic determinant or epitope of TdT. These monoclonal antibodies specifically recognize TdT in a wide variety of mammalian cells, including human, mouse, rat, rabbit and bovine origin. Three particular antibodies cross-react with the same epitope on TdT, and a fourth particular monoclonal antibody reacts with a distinct epitope, as determined by competitive displacement assay. These monoclonal antibodies bind to human and calf TdT, as well as extracts of rabbit, mouse and rat thymus which contain TdT-positive cells. However, these same antibodies do not bind to murine spleen which does not contain TdT-positive cells. These and other similar studies with the CID monoclonal antibodies to TdT have shown their specificity for TdT despite the species of origin and free of non-specific binding to other antigens.
These TdT monoclonal antibodies are disclosed in the co-pending patent application of R. Graham Smith, Ser. No. 802,039, filed Nov. 26, 1985 for "Monoclonal Antibodies To A Broad Range of Mammalian Terminal Deoxynucleotidyl Transferases."
We have encountered non-specific binding or staining of the cytoplasm and nucleoplasm of TdT negative cells in human peripheral blood with CID TdT specific monoclonal antibodies as well as with other commercially available TdT monoclonal antibodies. In order to develop clinically relevant analyses and enumerations of TdT positive cells in a peripheral blood sample, the nature of this non-specific binding phenomenon was sought. The assay protocol for staining for the presence of TdT was analyzed as a part of this process. The assay procedure includes the fixing of the cells being assayed in order to enable the conjugated TdT monoclonal antibody to penetrate the cytoplasm of the cell for staining the cell's nucleus for TdT.
We therefore analyzed all known fixatives that could be used for opening up large holes in the cell membrane for antibodies against TdT to enter without destroying the TdT molecule in the cell nucleus sought to be detected. A literature review revealed that other workers in the field experienced the same non-specific binding phenomenon using a two step or indirect prodecure. The nature of this binding was entirely independent of fixatives as well as the specificity of the antibodies employed in the staining process. For example, in the first step, a primary unlabelled antibody developed against TdT for staining the fixed cells for the presence of TdT was employed. A second step followed which involved adding an antibody specific for the immunoglobulin class of molecules of the species used to develop antibodies to TdT. Thus, in this indirect procedure, if the primary unlabelled antibody is a rabbit antibody against TdT, then the developing reagent carrying the fluorescent dye is a goat antibody to rabbit immunoglobulin. Therefore, the rabbit antibody to TdT binds to the TdT in the nucleaus of the fixed cell followed by the goat antibody to the rabbit antibody binding to it. Since the goat antibody carries the dye, we can now visualize the staining of TdT in the fixed cells by that rabbit antibody. However, the problem arose in this indirect technique, that if the goat antibody to rabbit immunoglobulin was applied to TdT negative or positive cells, all cells stained positively. Note, that the specificity has little or no relationship to cells staining with goat anti-rabbit immunoglobulin since it is human cells being stained and no circulating rabbit antibodies could be expected to be found in the human cells. Furthermore, no circulating rabbit antibodies could be expected to be found in the human patient samples being analyzed.
An incomplete but major aid in alleviating the nonspecific binding of the dye carrying goat antibody was to flood the fixed, TdT antibody stained sample with IgG which had no known specificity therein blocking the non-specific binding of dye carrying goat-anti-rabbit antiserum.
The conclusion drawn from these observations was that in order to develop a specific assay for TdT in fixed cells using a direct staining procedure as will be described herein, a blocking agent for the non-specific binding of monoclonal antibodies specific for TdT would be required. We therefore undertook the definition of the non-specific binding mechanisms which would account for background staining in TdT analyses. It was noted that af low cytometer instrument was uanble to differentiate between cytoplasmic versus nuclear TdT staining with sufficient accuracy to provide analyses from which the proper clinical conclusions could be drawn.
We have developed a unique chemical blocking agent which substantially prevents such non-specific binding of the TdT monoclonal antibody in the cytoplasm of the cells being assayed using a conjugated or tagged TdT monoclonal antibody. The chemical blocking agent is a phosphoprotein called casein and is specifically found in milk. The casein that effectively blocks non-specific binding of anti-TdT monoclonal antibodies may be derived from a variety of sources. Furthermore, a crude non-fat milk product also has been found to be effective as a blocking agent which contains casein. Further, the blocking agent is effective in an immunoassay even where a non-conjugated monoclonal antibody is utilized.